The thioesterases are substantially involved in the production of fatty acids in plant organisms. With respect to compartments the fatty acid and triacylglyceride biosynthesis can be viewed as separate biosynthesis. In view of the end product, they can be viewed as a single biosynthetic pathway. De novo biosyntheses of fatty acids is taking place in the plastids and is catalyzed by three enzymes or enzyme systems respectively, acetyl-coA Carboxylase (ACCase), the fatty acid synthase (FAS), and the acyl-ACP!-thioesterase (TE).
In most organisms the end products of these reactive pathways are either palmitic acid (C.sub.16:0), stearic acid (C.sub.18:0) and, after desaturation oleic acid (.DELTA.9C.sub.18:1). The acyl-ACP!-thioesterase (TE) flnctions in the determination of the length of the chain.
In contrast, triacylglyceride biosynthesis takes places at the endoplasmic reticulum in the cytoplasm via the so-called "Kennedy Pathway" from glycerin-3-phosphate, which is probably provided as a result of the activity of glycerin-3-phosphate dehydrogenase (G3P-DH), and fatty acids, which occur as acyl-coA substrates.
In animal systems (e.g. the rat), the acyl-ACP!-thioesterase is an integral part of the FASI and is responsible for the termination of the fatty acid biosynthesis there. A second acyl-ACP!-thioesterase (TEII), which is expressed in specific tissues, is responsible for the early termination of chain elongation in the milk producing glands of the rat breast, and causes the release of C.sub.10:0 and C.sub.12:0 fatty acids. Expression of this TEII in mouse fibroblasts resulted in the formation of these middle chain fatty acids in these cells. It is therefore concluded, that this enzyme is significantly involved in the termination of chain length. (S. A. Bayley et al., Bio/Technology 6, p. 1219-1221 (1988)).
Acyl-ACP!-thioesterases were also purified from plants, and analyzed for their activity. Acyl-ACP!-thioesterases with preference for the hydrolysis of long chain acyl-ACP! compounds were isolated from Carthamus tinctorius (T. A. McKeon et al., J. Biol. Chem. 257, p. 12141-12147 (1982)), Cucurbita moschata (H. Imai et al., Plant Mol. Biol. 20, p. 199-206 (1992)), and Brassica napus (A. Hellyer et al., Plant Mol. Biol. 20, p. 763-780 (1992)). Corresponding cDNAs have been isolated already from Carthamus tinctorius (D. S. Knutzon et al., Plant Physiol. 100, p. 1751-1758 (1992)) and Brassica napus (E. S. Loader et al., Plant Mol. Biol. 23, p. 769-778 (1993)). Another TE with specificity for the hydrolysis of C.sub.12:0 -ACP! has been isolated from Umbellularia Californica (California Laurel), and was separated from the activity of a C.sub.18:0 -ACP! specific TE (M. R. Pollard et al., Art. Biochem. Biophys. 284, p. 306-312 (1991)). In Cuphea lanceolata, the activity of a middle and a long chain-specific TE were detected as well (P. Dormann et al., Planta 189, p. 425-432 (1993)).
An only partially purified enzyme preparation of a C.sub.10:0 specific acyl-ACP!-thioesterase from Cuphea hookeriana is described in WO 91/16421. As measurements of the hydrolysis activities of the enzyme shows against various substrates, it contains significant amounts of activity which are not C10:0 specific.
For the TE from Umbellularia Californica a cDNA was isolated which codes for a middle chain-specific acyl-ACP!-thioesterase. This TE caused the formation of middle chain fatty acids in seeds of transgenic Arabidopsis thaliana and B. napus plants, in particular lauric acid (C12:0) and in small amounts myristic acid (C14:0); (T. A. Voelker et al., Science 257, p. 72-74 (1992) and H. M. Davies and T. A. Voelker in Murata, N. and C. Somerville (editors): Current Topics in Plant Physiology: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, Vol 9, p. 133-137; American Society of Plant Physiologists, Rockville (1993)).
There is an increasing demand on the supply of middle chain fatty acids, e.g. capric acid (C.sub.10:0), which can be used in industry as softeners, lubricants, pesticides, tensides, cosmetics, etc. One possibility to make these fatty acids available is in the isolation (extraction) of fatty acids from plants which show especially high contents of these fatty acids. The increase of content of middle chain fatty acids was achieved only to a limited extent by the classic method, which is the breeding of plants which produce elevated levels of these fatty acids.
Therefore it is the goal of this invention to provide genes or DNA sequences, which can be used to improve the yield of oils and the production of middle chain fatty acids in plants, which are not capable of producing these fatty acids themselves or only in small amounts.